Natural human Clara Cell 10 kDa protein (CC10), also known as uteroglobin, Clara cell 16 kDa protein (CC16), Clara cell secretory protein (CCSP), blastokinin, urine protein-1, and secretoglobin 1A1 (SCGB1A1), is one of a family of related proteins called secretoglobins believed to exist in all vertebrate animals. There are two additional secretoglobins that are also expressed at very high levels in the respiratory tract, called SCGB3A1 and SCGB3A2 (Porter, 2002). These three proteins; SCGB1A1, SCGB3A1, and SCGB3A2, are herein referred to as “respiratory secretoglobins.” Table 1 shows Genebank loci and amino acid sequences for each respiratory secretoglobin.
TABLE 1Respiratory secretoglobin proteinsGenebankProteinlocusAmino acid sequenceSCGB1A1BC004481EICPSFQRVIETLLMDTPSSYEAAMELFSPD(CC10)QDMREAGAQLKKLVDTLPQKPRESIIKLMEKIAQSSLCN (SEQ ID NO: 11) SCGB3A1NP_443095AAFLVGSAKPVAQPVAALESAAEAGAGTLANPLGTLNPLKLLLSSLGIPVNHLIEGSQKCVAELGPQAVGAVKALKALLGALTVFG(SEQ ID NO: 12) SCGB3A2AAQ89338ATAFLINKVPLPVDKLAPLPLDNILPFMDPLKLLLKTLGISVEHLVEGLRKCVNELGPEASEAVKKLLEALSHLV(SEQ ID NO: 3)
The primary source of respiratory secretoglobins in mammals is the pulmonary and tracheal epithelia, especially the non-ciliated bronchiolar airway epithelial cells (primarily Clara cells), and they are very abundant locally-produced proteins in the extracellular fluids of the adult lung. They are also secreted in the nasal epithelia. Thus, respiratory secretoglobins are highly expressed in both the upper and lower respiratory tracts; the upper respiratory tract includes the nasal passages and sinuses and the lower respiratory tract includes the trachea, bronchi, and alveoli of the lungs. A significant amount of respiratory secretoglobins are also present in serum and urine, which is largely derived from pulmonary sources. SCGB3A1 is also expressed in the stomach, heart, small intestine, uterine and mammary glands, and SCGB3A2 is expressed at a low level in the thyroid (Porter, 2002). CC10 is also produced by reproductive tissues (uterus, seminal vesicles), exocrine glands (prostate, mammary gland, pancreas), endocrine glands (thyroid, pituitary, adrenal, and ovary) and by the thymus and spleen (Mukherjee, 1999; Mukherjee, 2007). The major recoverable form of human CC10 in vivo is a homodimer, comprised of two identical 70 amino acid monomers, with an isoelectric point of 4.8. Its molecular weight is 15.8 kDa, although it migrates on SDS PAGE at an apparent molecular weight of about 10 kDa. The monomers are arranged in an antiparallel configuration, with the N-terminus of one adjacent to the C-terminus of the other, and in the fully-oxidized form of the dimer, the monomers are connected by two disulfide bonds (Mukherjee, 1999). However, the in vivo molecular form (monomer, dimer, or other complex) of SCGB3A2 in human samples has not yet been characterized. All three respiratory secretoglobins may be made by synthetic (Nicolas, 2005) or recombinant methods (Mantile, 1993), although there have been no reports to date describing the successful synthesis of human SCGB3A1 and SCGB3A2 and the biochemical characterization of these proteins in vitro.
CC10 is an anti-inflammatory and immunomodulatory protein that has been characterized with respect to various interactions with other proteins, receptors and cell types (reviewed in Mukherjee, 2007, Mukherjee, 1999, and Pilon, 2000). Lower levels of CC10 protein or mRNA have been found in various tissue and fluid samples for a number of clinical conditions characterized by some degree of inflammation including asthma (Lensmar, 2000; Shijubo, 1999; Van Vyve, 1995), pneumonia (Nomori, 1995), bronchiolitis obliterans (Nord, 2002), sarcoidosis (Shijubo, 2000), and in patients suffering from chronic rhinitis with recurrent sinusitis and nasal polyposis (Liu, 2004). Pulmonary epithelial cells, the body's primary source for endogenous CC10, are often adversely affected in these conditions, depleted or even ablated (Shijubo, 1999).
CC10 knockout (KO) mice have been important in characterizing the role of CC10 in pulmonary homeostasis, reproduction, and certain types of renal disease. There are two strains of CC10 KO mice, each with different genetic knockout constructs and different parental mouse strains. One knockout strain exhibits several extreme phenotypes, including systemic inflammation, poor reproductive capability (small litter sizes), and a lethal renal phenotype resembling human IgA nephropathy (Zhang, 1997; Zheng, 1999). The other knockout strain does not possess these extreme phenotypes and is more viable, enabling a greater number of experiments to be performed (Stripp, 1997). Both strains of CC10 KO mice share much greater sensitivity and significantly heightened inflammatory responses to pulmonary challenges in models of asthma, pulmonary fibrosis and carcinogenesis, bacterial and viral infections, and oxygen and ozone exposures (Plopper, 2006; Lee, 2006; Yang, 2004; Wang, 2003; Harrod, 2002; Chen, 2001; Wang, 2001; Hayashida, 2000; Harrod, 1998). Restoration of CC10 function in these knockout mice using recombinant human CC10 (rhCC10) has been shown to mitigate the exaggerated pulmonary inflammatory responses in short term challenge models with endpoints of up to 7 days (Chen, 2001; Wang, 2003). Most relevant to the invention, both strains share an airway epithelial phenotype characterized by significantly decreased numbers of Clara cells and associated structures called neuro-epithelial bodies (NEBs; Castro, 2000) or neuro-endocrine cell clusters (NECs; Hong, 2001; Reynolds, 2000), as identified by positive staining with calcitonin-gene related protein 1 (CGRP1). These 2-10 fold deficiencies in Clara cells and associated structures in the airways arise in the absence of any type of injury in these KO mice.
Premature infants who experience respiratory distress syndrome (RDS) are deficient in native CC10. In a clinical trial, a single dose of rhCC10 was administered on the day of birth and mediated potent short-term anti-inflammatory effects for 3-7 days in the lungs. Pharmacokinetic analyses showed that surplus CC10 was cleared within 48 hours of the single dose administered. Despite the anti-inflammatory effects, rhCC10 did not prevent development of neonatal bronchopulmonary dysplasia (BPD) (Levine, 2005), as defined by clinical parameters, including 1) opacity of chest X-ray at 28 days after birth or 2) use of supplemental oxygen at 36 weeks of postmenstrual age (PMA). Nor did rhCC10 reduce the time in the hospital or the number of days on the ventilator, despite the significant reductions in indices of pulmonary inflammation observed in tracheal aspirate fluids (TAF). There were no differences between the placebo, low dose and high dose treatment groups the 12 month endpoint, as stated in Levine et al. (2005).
Premature infants with BPD are predisposed towards experiencing frequent and severe respiratory exacerbations and their re-hospitalization rates in the first 1-2 years of life are high. Severe respiratory exacerbations are characterized by shortness of breath, labored breathing, nasal and chest congestion, overproduction of mucus, and sometimes respiratory distress. Severe respiratory exacerbations occur when patients encounter environmental exposures and infections through inhalation of dust, smoke, allergens, pollutants, chemicals, bacteria, fungi, and viruses.
Many types of patients with chronic diseases of the respiratory, gastrointestinal, urogenital tracts are susceptible to severe exacerbations when exposed to an environmental trigger. Likewise, patients with immunologic diseases, including autoimmune and allergic diseases, are also susceptible to severe exacerbations when exposed to an environmental trigger. Severe or acute exacerbations are considered frequent when they occur more than 3 times per year in a patient. Even patients who do not have a chronic disease, but who experience acute lung injury (ALI), are susceptible to frequent and severe acute respiratory episodes, resembling severe respiratory exacerbations, following the injury. Environmental irritants that trigger exacerbations include, but are not limited to, dust, particulates, smoke, allergens, pollutants, chemicals, contaminants, bacteria, fungi, and viruses may be inhaled, ingested, swallowed, absorbed through the skin, or otherwise come in contact topically with a wet mucosal surface of the patient's body.